The present invention relates to a polypeptide of N-acetylglucosamine-6-O-sulfotransferase and a DNA having a nucleotide sequence encoding the polypeptide.
The closest prior art is described below.
It has been described in Biochem. J., 319, 209-216 (1996) and J. Biol. Chem. 272, 29493-29501 (1997) that rat and human microsome fractions had an N-acetylglucosamine-6-O-sulfotransferase activity. However, there has been so far no report about isolation and identification of a polypeptide of N-acetylglucosamine-6-O-sulfotransferase. A DNA encoding this polypeptide has not also been known.
Once a polypeptide of N-acetylglucosamine-6-O-sulfotransferase is obtained, it can be used for synthesis of sugar chains such as GlyCAM-1 that is a ligand of L-selectin (which is involved in homing of lymphocytes and rolling of leukocytes occurring at the early stage of inflammation). A DNA encoding this polypeptide would be expected to be used for large scale production of the polypeptide, or artificial synthesis of GlyCAM-1 (having a structure of NeuAcxcex12-3Galxcex21-4(Fucxcex11-3) (SO4-6)GlcNAc-) by using transformants which harbors the DNA.
An object of the present invention is to provide a polypeptide of N-acetylglucosamine-6-O-sulfotransferase and a DNA encoding the polypeptide.
The present inventors have succeeded in cloning a DNA encoding a polypeptide of N-acetylglucosamine-6-O-sulfotransferase, having an activity to specifically transfer a sulfate group to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of GlcNAcxcex21-3Galxcex21-4GlcNAc, wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage. The inventors have also confirmed that the polypeptide of N-acetylglucosamine-6-O-sulfotransferase was expressed by the DNA and identified the polypeptide, thereby completing the present invention.
The present invention provides a polypeptide of (a) or (b) below (hereinafter sometimes referred to as xe2x80x9cthe polypeptide of the present inventionxe2x80x9d):
(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAcxcex21-3Galxcex21-4GlcNAcxe2x80x83xe2x80x83(I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage.
The polypeptide of the present invention also includes a polypeptide of (a) or (b) below:
(a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAcxcex21-3Galxcex21-4GlcNAcxe2x80x83xe2x80x83(I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage.
The polypeptide of the present invention also includes a polypeptide having the following properties:
(1) Action: a sulfate group is transferred from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAcxcex21-3Galxcex21-4GlcNAcxe2x80x83xe2x80x83(I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage;
(2) Substrate specificity: sulfate group is not transferred to chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, desulfated keratan sulfate, CDSNS-heparin, mucin from porcine stomach, mucin from bovine submaxillary gland, and an oligosaccharide represented by the formula II:
xe2x80x83Galxcex21-4GlcNAcxcex21-3Galxcex21-4GlcNAcxe2x80x83xe2x80x83(II)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage; and
(3) N-terminal amino acid sequence comprises an amino acid sequence represented by amino acid numbers 1 to 48 in SEQ ID NO: 2.
The present invention provides a DNA encoding a polypeptide of (a) or (b) below (hereinafter sometimes referred to as xe2x80x9cthe DNA of the present inventionxe2x80x9d):
(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAcxcex21-3Galxcex21-4GlcNAcxe2x80x83xe2x80x83(I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage.
The DNA of the present invention also includes a DNA encoding a polypeptide of (a) or (b) below:
(a) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 4; or
(b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a sulfate group from a sulfate group donor to a hydroxyl group at 6 position of an N-acetylglucosamine residue located at a non-reducing end of an oligosaccharide represented by the formula I:
GlcNAcxcex21-3Galxcex21-4GlcNAcxe2x80x83xe2x80x83(I)
wherein GlcNAc represents an N-acetylglucosamine residue, Gal represents a galactose residue, xcex21-3 represents a xcex21-3 glycosidic linkage, and xcex21-4 represents a xcex21-4 glycosidic linkage.
The DNA of the present invention is preferably a DNA comprising a nucleotide sequence represented by nucleotide numbers 470 to 1918 in SEQ ID NO: 1 and a DNA comprising a nucleotide sequence represented by nucleotide numbers 390 to 1841 in SEQ ID NO: 3.
The present invention also provides a method of producing a sulfated sugar (hereinafter sometimes referred to as xe2x80x9cthe producing method 1 of the present inventionxe2x80x9d) represented by the formula III:
xe2x80x83(SO4-6)GlcNAc-Rxe2x80x83xe2x80x83(III)
wherein GlcNAc represents an N-acetylglucosamine residue; SO4-6 means that a hydroxyl group at 6 position is sulfated, and -R represents a hydrogen atom or a sugar residue bonded by a glycosidic linkage,
which comprises a step of reacting the above-described polypeptide with a sugar chain represented by the formula IV:
GlcNAc-Rxe2x80x83xe2x80x83(IV)
wherein GlcNAc represents an N-acetylglucosamine residue, and -R represents a hydrogen atom or a sugar residue bonded by a glycosidic linkage.
Furthermore, the present invention provides a method of producing a sulfated sugar (hereinafter sometimes referred to as xe2x80x9cthe producing method 2 of the present inventionxe2x80x9d), which comprises a step of transfecting a cell with the DNA of the present invention into and then culturing the cell. A preferred embodiment of this method is a method of transfecting a cell with the DNA of the present invention and a cDNA encoding fucosyltransferase concurrently.
The present invention further provides an antibody that reacts with 6-sulfated sialyl Lewis X but does not react with 6xe2x80x2-sulfated sialyl Lewis X, 6,6xe2x80x2-bis-sulfated sialyl Lewis X, 6-sulfated Lewis X and Lewis X.
The present invention still further provides an antibody that specifically reacts with a sugar chain represented by the following formula:
Galxcex21-4 (Fucxcex11-3) (SO4-6) GlcNAc
The present invention also provides an antibody that reacts with a sugar chain represented by the following formula:
NeuAcxcex12-3Galxcex21-4(SO4-6)GlcNAc.